The present invention relates to the use of LAG-3 and CD4, and in a more general way, the use of MHC class II ligands or MHC class II-like ligands as adjuvants for vaccines, in order to boost an antigen specific immune response, as well as the use of LAG-3 as a therapeutical agent in cancer immunotherapy.
It is now recognized that the proteins encoded by MHC Class II region are involved in many aspects of immune recognition, including the interaction between different lymphoid cells such as lymphocytes and antigen presenting cells. Different observations have also shown that other mechanisms which do not take place via CD4 participate in the effector function of T helper lymphocytes.
The lymphocyte activation gene 3 (LAG-3) expressed in human CD4+ and CD8+ activated T-cells as well as in activated NK cells encodes a 503 amino-acids (aa) type I membrane protein with four extracellular immunoglobulin superfamily (IgSF) domains (1) and is a ligand for MHC class II molecules (2). Analysis of this sequence revealed notable patches of identity with stretches of aminoacids sequences found at the corresponding positions in CD4, although the overall aminoacids sequence homology with human CD4 is barely above background level (approximately 20% sequence identity). There are also some internal sequence homologies in the LAG-3 molecule between domains 1 (D1) and 3 (D3) as well as between domains 2 (D2) and 4 (D4) suggesting that LAG-3 has evolved like CD4 by gene duplication from a preexisting 2 IgSF structure (1). In addition, LAG-3 and CD4 genes are located in a very close proximity on the distal part of the short arm of chromosome 12 (3). LAG-3 and CD4 can therefore be regarded as evolutionary xe2x80x9cfirst cousinsxe2x80x9d within the IgSF (2).
Like CD4, hLAG-3 is composed of lg like ectodomains with a WxC signature motif in domains 2 and 4; however a difference with CD4 is the presence of an extraloop sequence in domain 1 (recognized by the 17B4mAb) 30 and an intracytoplasmic proline rich motif (EP repeats) in human LAG-3 (hLAG-3). Recently, murine lymphocyte activation gene 3 (mLAG-3) was cloned and approximately 70% of homology was found with hLAG-3, with the same proline rich motif in the intracytoplasmic tail.
Antigen specific stimulation of CD4+ T-cell clones in the presence of anti-LAG-3 mAb leads to extended proliferation and cytokines production (5). It has been suggested a regulatory role of hLAG-3 on CD4+ T lymphocyte activated, by cross-linking MHC class II molecules expressed on T-cells with LAG-3 lg fusion proteins (6). LAG-3 MHC class II interaction inhibits signals through MHC class II molecules expressed on CD4+ T-cells (decrease of proliferation and cytokines production), suggesting that both LAG-3 and MHC class II are effector molecules for the down-regulation of T helper cell mediated immune responses. The hLAG-3 lg fusion protein was found to bind xenogenic MHC class II molecules (murine and monkey). In addition, the mLAG-3 has been proposed to transduce a positive signal in effector cells, since transgenic mice with a LAG-3 null mutation have a defect in the NK cell compartment (7).
Mouse tumor cell lines engineered to express membrane (B7.1, B7.2. CD95L, . . .) or secreted molecules (IL-2, IL-12,. . .) are often used to investigate immune responses or antitumor effects. This approach implies that many tumor cells are potentially antigenic (9), and become immunogenic when they express molecules. Experimental mouse tumors are classified as intrinsically immunogenic when, after a single injection into syngenic mice as nonreplicating cell vaccines, they elicit a protective immune response against a subsequent lethal challenge. Tumors that do not retain this residual immunogenicity are defined as poorly immunogenic or nonimmunogenic.
Antitumor immune responses are mediated primarily by T-cells (12). Recent studies have implicated a deficit in efficient antigen presentation and T-cell priming as being problematic for the practical implementation of an ideal tumor vaccine. Indeed, it has been demonstrated that transfecting tumor cells with genes coding for various cytokines, such as IL-2, IL-4, IL-12 or GM-CSF or genes coding for co-stimulatory molecules such as B7 not only led to primary rejection of the modified cells but often elicited protective immunity against subsequent challenge with unmodified tumor cells (13).
Professional antigen presenting cells (APCs) are capable of taking up, processing and presenting antigen to T-cells in the context of co-stimulatory signals required for T-cell activation, leading to optimal antigen presentation. In particular, it is well established that MHC class II+ dendritic cells (DCs) play a crucial role in processing and presenting antigens to the immune system. The inventors hypothesized that tumor immunogenicity would be increased if tumor could be modified to directly trigger host APCs such as macrophages and dendritic cells. Indeed, it has been reported that cross-linking of MHC class II molecules specifically expressed by such cells, using mAb or superantigens, transduces signals resulting in TNFxcex1 and IL-12 production (14, 15). They had previously reported that Lymphocyte Activation Gene-3 (LAG-3), which is embedded in the CD4 locus (1, 16), encodes a protein that binds human and murine MHC class II molecules with higher affinities than CD4 (17, 6).
The inventors of the instant application have investigated whether hLAG-3, human CD4 (hCD4) and mLAG-3 expression on three MHC class II-mouse tumors (the poorly immunogenic sarcoma MCA 205 and the nonimmunogenic TSIA+RENCA adenocarcinoma) can mediate an immune response so as to reject mouse tumor and can induce systemic immunity.
As a result, they have discovered that human or murine LAG-3, whether expressed as membrane proteins in solid tumor cell lines or inoculated into mice as a soluble protein induced a potent immunity against highly malignant murine tumors. The immunity was T-cell dependent and antigen-specific.
They have further investigated the role of CD4 and found that human CD4 (hCD4) also induced a systemic antitumor response.
The induced immunity has been found to be T-cell mediated, since the same antitumor response was obtained with Nude mice lacking T-lymphocytes.
The antitumor effect was still found when using different tumor cell lines exhibiting different intrinsic immunogenicity as well as different strains of mice expressing different MHC genes.
Furthermore, the hLAG-3 and hCD4 induced effects were observed when tumor cell lines expressing hLAG-3 or hCD4 were injected at a distant site from the initial inoculation site of the wild-type tumor cell lines.
Furthermore, systemic administration of soluble hLAG-3 directly induces an inhibition of in vivo tumor growth.
All the aforementioned results demonstrate that LAG-3 and CD4 are able to elicit an antigen specific T-cell mediated immune response and may be useful as a tool in immunotherapy, in order to prevent the occurrence of a cancer among populations at risk or more generally in any immunotherapy involving an antigen-specific T-cell mediated immune response, and that LAG-3 is further useful as a tool for inhibiting in vivo tumor growth.
The inventors have further demonstrated that soluble LAG-3 when administered together with an antigen against which an immune response is sought, was able to work as an adjuvant for a vaccine.
This role can be explained by an improved presentation of the antigen by professional APCs (dendritic cells and macrophages) located underneath the skin and triggered via MHC class II.
Accordingly, since induction of a CD8+ T-cell immunity is involved in viral (e.g. AIDS, hepatitis and herpes) and intra-cellular parasitic and bacterial (e.g. leprosy tuberculosis) infections and cancer, LAG-3 will be particularly useful for therapeutic vaccination against the pathogen agents involved in these diseases as well as in cancer treatment.
According to one of its aspects, the present invention relates to the use of a MHC class II ligand or a MHC class II-like ligand for the manufacture of a medicament for preventing or treating pathological conditions involving an antigen specific immune response, preferably an antigen-specific T-cell mediated immune response.
In a first embodiment, the MHC class II binding molecule is LAG-3 as well as derivatives thereof, able to bind HC class ligand of LAG-3.
By derivatives of LAG-3, in the sense of the present invention, there are meant mutants, variants and fragments of LAG-3 namely soluble fragments of LAG-3 provided that they maintain the ability of LAG-3 to bind MHC class II molecules.
Thus, the following forms of LAG-3 may be used:
the whole LAG-3 protein,
a soluble polypeptide fragment thereof consisting of at least one of the four immunoglobulin extracellular domains, namely the soluble part of LAG-3 comprised of the extracellular region stretching from the aminoacid 23 to the aminoacid 448 of the LAG-3 sequence disclosed in French Patent application FR 90 00 126,
a fragment of LAG-3 consisting of substantially all of the first and second domains
a fragment of LAG-3 consisting of substantially all of the first is and second domains or all of the four domains, such as defined in WO 95/30750, such as
a mutant form of soluble LAG-3 or a fragment thereof comprising the D1 and D2 extracellular domains and consisting of:
a substitution of an aminoacid at one of the following positions:
position 73 where ARG is substituted with GLU,
position 75 where ARG is substituted with ALA or GLU,
position 76 where ARG is substituted with GLU, or a combination of two or more of those substitutions,
a substitution of an aminoacid at one of the following positions:
position 30 where ASP is substituted with ALA;
position 56 where HIS is substituted with ALA;
position 77 where TYR is substituted with PHE;
position 88 where ARG is substituted with ALA;
position 103 where ARG is substituted with ALA;
position 109 where ASP is substituted with GLU
position 115 where ARG is substituted with ALA;
or a deletion of the region comprised between the position 54 and the position 66,
or a combination of two or more of those substitutions.
Those mutants are described in PNAS, June 1997 (4)
or a physiological variant of LAG-3 comprised of a soluble 52 kD protein containing D1, D2 and D3.
According to a second embodiment, the MHC class II binding protein is CD4 or a derivative thereof able to bind the MHC class II ligand of CD4.
The derivatives of CD4 are such as defined for the derivatives of LAG-3. They are namely mutants, variants and fragments of CD4 namely soluble fragments of CD4 provided that they maintain the ability of CD4 to bind MHC class II molecules.
LAG-3 and CD4, namely hLAG-3 and hCD4 or the derivatives thereof such as defined above may be administered as recombinant moieties expressing said molecules, for example transfected cells or recombinant viruses.
The present invention relates also to tumor cells transfected with a DNA coding for at least one MHC class II ligand, such as CD4 or LAG-3 or derivatives thereof.
A further object of the instant invention is also the use of cells, like tumor cells, transfected with a DNA coding for at least one MHC class II ligand, such as CD4 or LAG-3 or derivatives thereof for the manufacture of a medicament, preferably a medicament for preventing or treating pathological conditions involving an antigen specific immune response like an antigen specific T-cell mediated immune response or for treating pathological disorder like cancers.
The transfected cells are preferably mammal cells and in particular mammal tumor cells.
According to one of its aspects, the present invention relates to a process for preparing cells transfected with a DNA coding for at least one MHC class II ligand, such as CD4 or LAG-3 or derivatives thereof comprising the steps consisting of removing cells from a patient, transfecting said cells with a DNA coding for at least one MHC class II-like ligand, such as CD4 or LAG-3 or derivatives thereof and recovering the so-transfected cells.
For the preparation of tumor cells according to the invention, this process will be reproduced on tumor cells removed from a patient.
However, according to a preferred embodiment, the MHC class II binding protein, namely CD4 or LAG-3 or the derivative thereof, is administered in a free form, namely in a soluble form by inoculating them systemically, for example as an s.c, i.m or i.v injection.
The medicament according to the invention may be used as a vaccine to prevent disorders associated with an antigen specific immune response, preferably a T-cell mediated immune response.
To that end, it is administered in a suitable vehicle together with one or several antigen(s) against which an immune response is sought. The antigen may be an inactivated or attenuated infectious agent or a purified antigen, eventually obtained by protein recombinant procedures, such as an antigen of an infectious agent or a tumor antigen, which preferably are able to elicit a T-cell mediated immune response.
The vaccine may be used to prevent a subject against an infectious disease, such as a viral, bacterial or parasitic disease wherein the infectious agent elicits an antigen specific immune response, preferably a T-cell mediated immune response.
The vaccine may also be used for treating patient against an infectious disease such as mentioned hereabove, involving a T-cell mediated immune response, namely a CD8+ T-cell mediated immune response.
Examples of diseases requiring a boost of an existing T-cell mediated immunity are provided in the following table.
In such cases, the antigen is chopped in the cells and the corresponding peptides loaded into MHC class I molecules and presented at the surface of the cells where there are recognized by CD8+ cells. The results of the inventors showing that LAG-3lg molecules induce efficient T-cell response in animals and stimulate immature dendritic and monocytes in vitro strongly suggest that LAG-3 is a natural T-cell adjuvant in situations where it can cross-link MHC class II molecules in professional APCs.
The vaccine may also be used to prevent a subject against cancer, either solid tumor cancer or leukemia.
The vaccine may further be used for treating a patient against cancer.
In that case, the MHC class II binding protein namely LAG-3 or CD4 is administered to a subject either subcutaneously, intradermically or as a nasal spray together with one or several antigens able to elicit an immune response, preferably a T-cell mediated immune response. The antigen may be a peptide, a lipopeptide, a recombinant protein or DNA coding for these antigens.
The anti-cancer vaccine may be inoculated to populations at risks identified by their genotype (preventive vaccine) or to patients (therapeutic vaccine) bearing a tumor or at high risk of relapse following surgery.
Whether the vaccine is used as a conventional vaccine (preventive) or a therapeutic vaccine, it may be administered as a xe2x80x9cnakedxe2x80x9d plasmid (19) incorporating a DNA sequence encoding LAG-3 or CD4, preferably under the control of a strong promoter.
The plasmid preferably also contains DNA encoding the antigen against which an immune response is sought.
A further object of the instant invention is thus a pharmaceutical composition comprising an effective amount of a MHC class It ligand in combination with an effective amount of an antigen able to stimulate the immune system, preferably via a T-cell response.
In still another aspect, the present invention relates to the use of LAG-3 as a medicament for anti-cancer immunotherapy in patients bearing a cancerous tumor.
In that case, LAG-3 is administered preferably as a free LAG-3 protein or a derivative thereof in a pharmaceutically acceptable vehicle, preferably a soluble derivative such as defined previously.
LAG-3 may be administered as an intratumoral injection or systemic injection, for example s.c, i.v or i.m.
A further object of the present invention relates to a method for tumor gene therapy comprising the steps consisting of removing a portion of a patient tumor cells, transfecting said cells with a DNA coding for at least one MHC class II ligand, such as CD4 or LAG-3 or derivatives thereof and re-introducing the so-transfected cells into the patient.
The following examples demonstrate the activity of LAG-3 and CD4 in the prevention or treatment of pathological conditions involving a T-cell mediated immune response.